Organic phosphates act as both substrates and allosteric effectors for a host of protein systems. While elegant studies by numerous laboratories have established the physiological significance of these organophosphate effectors, the determination of binding constants and their respective energies which regulate these important interactions have not been determined. Since there is a wealth of structural and functional information on hemoglobin, it is, perhaps, the best model protein system to study the interactions between organophosphates and macromolecules. Moreover, the involvement of other ligands (e.g., C1-, HCO3-, H+, etc.) make hemoglobin an excellent model for the study of protein-ligand interactions in general. We propose to investigate the mode of binding between 2,3-diphosphoglycerate (DPG) and human hemoglobin as a function of pH, temperature, protein concentration, and oxygenation state of the protein. Our proposed research will delineate for the first time the following important features for the regulation of the hemoglobin system: (1) the detailed nature and mechanism of the interactions of hemoglobin with organic phosphates and other ligands, (2) the energies of interaction and energy states of the various molecular species involved, and (3) the regulatory constraints imposed by the various ionic species of DPG over the physiologically significant pH range. The elucidation of these energy states and the regulatory constraints will provide an important missing link in understanding the hemoglobin-ligand system. This is particularly important because it is these energies that provide the driving force for the regulation of hemoglobin function.